Characterization of a recombinant multifunctional glycoside hydrolase family 3 β-xylosidase/α-l-Arabinofuranosidase/β-glucosidase from Cellulosimicrobium cellulans sp. 21

Article abstract of DOI:10.1016/j.molcatb.2016.06.002

A multifunctional β-xylosidase/α-l-Arabinofuranosidase/β-glucosidase gene (ccxyl3a) belonging to glycoside hydrolase family 3 (GH3) was cloned from Cellulosimicrobium cellulans sp. 21 and expressed in Escherichia coli BL21 (DE3). The molecular mass of recombinant CcXyl3A was estimated to be approximately 95 kDa. With p-nitrophenyl-β-d-xyloside (pNPβXyl) as a substrate, the purified protein presented an optimal pH of 8.5 and an optimal temperature of 45 °C. Moreover, CcXyl3A was activated in the presence of the metals K⁺ and Na⁺. Purified CcXyl3A demonstrated multifunctional activities on pNPβXyl, p-nitrophenyl-β-d-glucoside (pNPβGlc), and p-nitrophenyl-α-l-Arabinofuranoside (pNPαAraf). The greatest catalytic activity were found on pNPβXyl followed by pNPαAraf and pNPβGlc, respectively. Using xylooligosaccharides as substrate, CcXyl3A completely hydrolyzed xylobiose, xylotriose, xylotetraose and xylohexaose, xylose was the sole product. In addition, CcXyl3A synergistically acted with Thermomyces lanuginosus xylanase in the degradation of beechwood xylan, released xyloses from intermediate xylooligosaccharides produced by T. lanuginosus xylanase. To date, this is the first report to demonstrate the cloning and characterization of a multifunctional GH3 enzyme in C. cellulans that may have applications in hemicellulose degradation.

Full text of DOI:10.1016/j.molcatb.2016.06.002

Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Characterization of a recombinant multifunctional glycoside
hydrolase family 3
␤
-xylosidase/␣-l-arabinofuranosidase/␤-glucosidase from
Cellulosimicrobium cellulans sp. 21
a
a
a
a
a
c
a,b,∗
Ye Yuan , Yanbo Hu , Han Zhang , Jiayi Leng , Fan Li , Xuesong Zhao , Juan Gao
,
a,∗
Yifa Zhou
a
Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University,
Changchun 130024, PR China
School of Biological Science and Technology, University of Jinan, Jinan 250022, PR China
b
c
College of Mining Industry, Liaoning Technical University, Fuxin 123000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A multifunctional ␤-xylosidase/␣-l-arabinofuranosidase/␤-glucosidase gene (ccxyl3a) belonging to gly-
coside hydrolase family 3 (GH3) was cloned from Cellulosimicrobium cellulans sp. 21 and expressed in
Escherichia coli BL21 (DE3). The molecular mass of recombinant CcXyl3A was estimated to be approxi-
mately 95 kDa. With p-nitrophenyl-␤-d-xyloside (pNP␤Xyl) as a substrate, the purified protein presented
Received 28 December 2015
Received in revised form 11 May 2016
Accepted 3 June 2016
Available online 10 June 2016
◦
an optimal pH of 8.5 and an optimal temperature of 45 C. Moreover, CcXyl3A was activated in the pres-
+
+
ence of the metals K and Na . Purified CcXyl3A demonstrated multifunctional activities on pNP␤Xyl,
p-nitrophenyl-␤-d-glucoside (pNP␤Glc), and p-nitrophenyl-␣-l-arabinofuranoside (pNP␣Araf). The
greatest catalytic activity were found on pNP␤Xyl followed by pNP␣Araf and pNP␤Glc, respectively.
Using xylooligosaccharides as substrate, CcXyl3A completely hydrolyzed xylobiose, xylotriose, xylote-
traose and xylohexaose, xylose was the sole product. In addition, CcXyl3A synergistically acted with
Thermomyces lanuginosus xylanase in the degradation of beechwood xylan, released xyloses from inter-
mediate xylooligosaccharides produced by T. lanuginosus xylanase. To date, this is the first report to
demonstrate the cloning and characterization of a multifunctional GH3 enzyme in C. cellulans that may
have applications in hemicellulose degradation.
Keywords:
Cellulosimicrobium cellulans
Cloning and expression
Glycoside hydrolase family 3
Substrate specificity
␤
-xylosidase/␣-l-arabinofuranosidase/␤-
glucosidase
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
as ␤-xylosidase/␣-l-arabinofuranosidase and ␤-glucosidase/␤-
xylosidase/␣-arabinosidase, which could hydrolyze a diverse range
of fibre substrates [5,6]. Identification and utilization of novel mul-
tifunctional hemicellulolytic enzymes is of potentially useful for
the conversion of hemicellulosic biomass into biofuels, as well as
for the preparation of other useful chemicals [7,8].
Hemicellulose is the second most common polysaccharides
in nature, including xylan, glucuronoxylan, arabinoxylan, gluco-
mannan, and xyloglucan. These polysaccharides contain pentoses
such as xylose and arabinose, and hexoses such as glucose
and mannose [1,2]. Owing to the heterogeneity in composi-
tion and structure, the complete degradation of hemicellulosic
biomass requires the synergetic actions of different enzymes
Glycoside hydrolase family
3
(GH3) is the main
GH family involving exoacting ␤-d-glucosidases, ␣-l-
arabinofuranosidases, and ␤-d-xylosidases (www.cazy.org).
The ␣-l-arabinofuranosidases and ␤-d-xylosidases from GH3
are involved in the degradation of hemicellulosic xylan and
arabinoxylan. Many of the GH3 enzymes have dual or broad
substrate specificities with respect to monosaccharide residues,
linkage position, and chain length of the substrate [9,10]. Previ-
ously, several bifunctional ␣-l-arabinosidases/␤-xylosidases and
multifunctional ␤-glucosidases/␤-xylosidases/␣-arabinosidases
belonging to GH family 3 have been characterized [11–13].
[
3,4]. It is reported that many microorganisms produced a serial
of divergent enzymes to degrade hemicellulose. In order to
achieve efficiently and completely degradation, some microorgan-
isms have evolved multifunctional hemicellulolytic enzymes, such
∗ Corresponding authors.
E-mail addresses: gaoj199@nenu.edu.cn (J. Gao), zhouyf383@nenu.edu.cn
(
Y. Zhou).
http://dx.doi.org/10.1016/j.molcatb.2016.06.002
381-1177/© 2016 Elsevier B.V. All rights reserved.
1

6
6
Y. Yuan et al. / Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
Cellulosimicrobium cellulans, previously designated as Oerskovia
gel [17]. Protein concentrations were determined by the method of
Bradford using bovine serum albumin (BSA) as a standard.
xanthineolytica, is an actinobacteria with yeast-lytic glucanases
activity [14,15]. Previously, C. cellulans was reported to pro-
duce hemicellulose-degradation enzymes in culture medium when
using corn cob as sole carbon source [16]. In this study, a novel mul-
tifunctional ␤-xylosidase/␣-l-arabinofuranosidase/␤-glucosidase
gene belonging to GH family 3 was cloned from C. cellulans sp.21,
the recombinant protein, CcXyl3A, was expressed, purified and the
enzymatic properties were investigated. Additionally, the utility of
CcXyl3A for the degradation of xylan was investigated by examin-
ing its enzymatic properties in combination with xylanase.
2.4. Enzymatic characterization
The specific activity measurement was carried out in 200 ␮l of
50 mM phosphate buffer (pH 8.0) containing 40 mM substrate and
◦
2 ␮g recombinant CcXyl3A. After incubated at 30 C for 10 min,
the reaction was stopped by adding 100 ␮l NaOH (0.2 M), and
the released p-nitrophenol was measured at 405 nm by BioTek
ELx808 microplate reader (Winooski, VT, USA) [18]. The reaction
mixture without enzyme was used as a blank. The enzymatic
activities of CcXyl3A on sophorose, laminaribiose, laminaritriose,
cellobiose, cellotriose, gentiobiose, xylobiose and xylotriose were
measured by determining the reducing sugers with dinitrosalicylic
acid (DNS) reagent at 520 nm [19]. One unit (U) of enzyme activ-
ity was defined as the amount of enzyme releasing 1 ␮mol/min of
p-nitrophenyl/reducing sugar under assay conditions.
2
. Materials and methods
2
.1. Strains and reagents
Strain C. cellulans sp. 21 was isolated from the soil in Changbai
Mountain (Jilin Province, China), and stored in China General
Microbiological Culture Collection Center (collection number
CGMCC 7587) [18]. E. coli BL21 (DE3) and pET-28a (+) were used
as host and expression vector, respectively (Novagen, Madison,
WI, USA). p-Nitrophenyl-␤-glucopyranoside (pNP␤Glc), pNP-␣-
glucopyranoside (pNP␣Glc), pNP-␤-galactopyranoside (pNP␤Gal),
pNP-␣-galactopyranoside (pNP␣Gal), pNP-␤-mannopyranoside
The effect of pH on CcXyl3A activity was determined at differ-
◦
ent pHs ranging from 2.0 to 10.5 at 30 C for 10 min using pNP␤Xyl
(40 mM) as a substrate. The pH stability was investigated under
standard assay conditions after incubation of the purified CcXyl3A
◦
for 24 h at 4 C in the buffers without substrate. The optimum tem-
perature was determined by measuring the enzymatic activity at
◦
optimal pH in the temperatures ranging from 20 to 90 C. Ther-
(
␤
(
pNP␤Man),
-xylopyranoside
pNP␣Araf) and T. Lanuginosus xylanase (X2753-10G) was from
pNP-␣-mannopyranoside
(pNP␣Man),
pNP-
mostability was performed by incubating the enzyme at different
(pNP␤Xyl), pNP-␣-l-arabinofuranoside
◦
temperatures (30–50 C) for up to 1 h. The residual activities were
◦
assayed using pNP␤Xyl as a substrate at 30 C for 10 min. The initial
Sigma (St. Louis, MO, USA). Genomic DNA isolation, DNA purifica-
tion, and plasmid isolation kits were from Tiangen Biotech (Beijing,
China). All of other chemicals and reagents were analytical grade.
activity was defined as 100%.
The effects of metals and other chemicals on CcXyl3A activity
were determined. CcXyl3A was pre-incubated in 20 mM Tris-HCl
buffer (pH 7.0) in the presence of metals or reagents (5 or 50 mM,
2
.2. Cloning and expression of ccxyl3a gene
◦
final concentration) at 30 C for 1 h. Then the residual activity
was determined using pNP␤Xyl as a substrate (final concentration
Genomic DNA of C. cellulans sp. 21 was isolated using Tian-
4
0 mM) as described before.
gen genomic DNA isolation kit. The primers having restriction
The effect of sugars (glucose, xylose and arabinose) on CcXyl3A
ꢀ
ꢀ
sites for NdeI (5 -GGAATTCCATATGACCGACGTCGTCCCTGCCGT-3 )
activity was carried out by pre-incubating CcXyl3A for 1 h at opti-
mal pH and temperature with 5, 10, 25, 50 or 100 mM of each sugar.
The retained activity of CcXyl3A was then measured with pNP␤Xyl
as a substrate and compared to that in the absence of sugar.
ꢀ
ꢀ
and BamHI (5 -CGGGATCCTCATCATGCGCGCTCCCCCGCCT-3 ) were
used for gene ccxyl3a amplification. PCR was performed using Phu-
◦
sion polymerase and the following program: 98 C for 30 s, 30 cycles
◦
◦
◦
of 98 C for 10 s, 68 C for 45 s, 72 C for 1 min, and final exten-
sion at 72 C for 10 min. The PCR product and pET-28a (+) were
◦
2.5. Determination of kinetic parameters
digested with NdeI and BamHI, the ccxyl3a gene was ligated with
pET-28a (+) to generate the recombinant plasmid pET28a-ccxyl3a.
For CcXyl3A expression, E. coli BL21 (DE3) cells harboring pET28a-
The kinetic parameters of CcXyl3A were determined by measur-
ing the initial reaction velocity at different substrate concentrations
ranging from 0.1 mM to 5 mM at optimal pH and temperature. The
Km and Vmax values were calculated from GraphPad Prism V5.
ccxyl3a were grown in 200 ml of LB broth with 50 ␮g/ml kanamycin
◦
at 37 C. When the OD
reached 0.5, the culture was induced
6
00nm
◦
with 0.5 mM IPTG and then grown for 20 h at 25 C.
2.6. Xylo-oligosaccharides degradation by CcXyl3A
2.3. Purification of recombinant CcXyl3A by Ni sepharose fastflow
column
The hydrolytic activity of CcXyl3A on xylo-oligosaccharides
was determined. The reaction mixture of 200 ␮l composed of
50 mM Na2HPO4-NaH2PO4 buffer (pH 8.0), 10 mg/ml xylobiose
(X2), xylotriose (X3) or xylo-oligosaccharides mixture (XOS-70,
Shandong Longli Co.) and 10 ␮g/ml purified CcXyl3A was incu-
◦
Cells were harvested by centrifugation at 4 C and 14,000g for
1
0 min, suspended in 20 ml of lysis buffer (0.1 M NaCl, 20 mM Tris-
HCl, pH 7.5) and then disrupted by sonication on ice (3 s pulse
with 3 s interval for 5 min). Cell debris were removed by cen-
trifugation (14,000g, 4 C, 30 min). The crude extract containing
CcXyl3A was loaded onto Ni sepharose fastflow column (GE health-
care). The column of 5 ml was pre-equilibrated by binding buffer
◦
bated at 30 C for 10 min. The hydrolytic products of xylobiose and
◦
xylotriose were determined by thin layer chromatography (TLC).
The TLC plates were developed using ethyl acetate/methanol/water
(16/6/1, v/v/v) as developing solvent. Products were visualized by
spraying the plates with sulphuric acid/ethanol (5/95, v/v) followed
(
10 mM imidazole, 0.1 M NaCl, 20 mM Tris-HCl, pH 7.5), washed
◦
with 20 ml of washing buffer (20 mM imidazole, 0.1 M NaCl, 20 mM
Tris-HCl, pH 7.5) and eluted with 20 ml of eluting buffer (250 mM
imidazole, 0.1 M NaCl, 20 mM Tris-HCl, pH 7.5). The active frac-
tions were pooled and dialyzed against 20 mM Tris-HCl buffer (pH
.5). The purified CcXyl3A was analyzed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) on 10% separating
by heating for 5 min at 105 C in an oven. The hydrolytic products
of xylo-oligosaccharides mixture were determined by Inertsil NH2
column (DIKMA 4.6 × 250 mm, 5 ␮m) connected to an HPLC sys-
tem (Shimadzu, Japan), eluted at the flow rate of 1.0 ml/min for
15 min with 65% acetonitrile (in distilled water, v/v), monitored by
the refractive index detector.
7

Y. Yuan et al. / Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
67
2.7. Synergistic action of CcXyl3A with xylanase during the
and BglX-V-Ara from Caulobacter crescentus (WP 010920890, 37%
hydrolysis of beechwood xylan
identity), which suggested that CcXyl3A is a novel trifunctional
␤-xylosidase/␣-l-arabinofuranosidase/␤-glucosidase among fam-
Hydrolysis of xylan was performed by incubating 10 mg/ml
beechwood xylan with T. lanuginosus xylanase (Xyn, final con-
centration 10 ␮g/ml), or 10 ␮g/ml CcXyl3A, or both in 50 mM
phosphate buffer (pH 8.0), at 37 C and 150 rpm for 8 h. The amount
of reducing sugars released from the reaction at various time points
ily 3 glycoside hydrolases.
3.2. Overexpression, purification, and molecular mass
◦
determination of CcXyl3A
(
0, 1, 2, 4, 8 h) was measured by DNS method, and comparing it
To systematically study the function of CcXyl3A, the gene was
cloned into the expression vector pET-28a and overexpressed in
E. coli. After induction with 0.5 mM IPTG at 25 C for 20 h, the recom-
to a standard curve of xylose. To identify the hydrolysis products,
aliquots of hydrolysate were removed at 0, 1, 2, 4 and 8 h, respec-
tively, and analyzed by TLC method as described before.
◦
binant enzyme CcXyl3A accounted for approximately 50% of the
total soluble cellular protein (data not shown). The concentration
of total protein was 167.5 mg/l culture and the specific activity of
recombinant CcXyl3A was 1.9 U/mg. CcXyl3A was purified by Ni
sepharose fastflow column with a final purification of 2.33-fold,
a yield of 69.4%, and a specific activity of 4.42 U/mg. The detailed
purification results from a representative 200-ml culture are sum-
marized in Table 1. The expressed protein analyzed by SDS-PAGE
showed a single band after one-step affinity chromatography, with
a molecular mass of approximately 95 kDa (Fig. 2). The molecu-
lar weight of CcXyl3A is similar to that of some GH3 bifunctional
or multifunctional enzymes, such as Bgxa1 (81.7 kDa), RubGX1
2
.8. Nucleotide sequence accession numbers
The sequences for the 16 S rRNA and CcXyl3A genes from C. cel-
lulans sp. 21 were deposited in GenBank under accession numbers
KR349463 and KT833270, respectively.
3
. Results and discussion
3
.1. Sequence analysis
(
80 kDa) and BglX-V-Ara (95 kDa) [11–13]. Compared with other
GH3 enzymes such as ␤-xylosidase, ␣-l-arabinofuranosidase
non-secretory proteins which are difficult to be purified, recom-
binant CcXyl3A can be easily purified, involving in only one-step
affinity chromatography.
and ␤-glucosidase are proved to be key enzymes for the degra-
dation of hemicellulosic biomass. C. cellulans was reported to
produce hemicellulose-degradation enzymes in culture medium
when using corn cob as sole carbon source [16]. However, the genes
encoding for GH3 enzymes in C. cellulans have not yet been well
characterized. In this study, a GH3 gene ccxyl3a from C. cellulans
was cloned and overexpressed in E. coli. Gene ccxyl3a consisted of
3.3. Characterization of recombinant CcXyl3A
The effect of pH on CcXyl3A activity was examined ranging from
2.0 to 10.5 with pNP␤Xyl as a substrate. Our results showed that
the maximum activity was observed at pH 8.5 (Fig. 3a), which was
similar to that of the GH3 multifunctional enzyme BgxA1 (optimal
pH 8.5 for pNP␤Xyl), but different from BglX-V-Ara, the optimal
pH of which was observed at 6.0. CcXyl3A was stable within the
pH range from 6.0 to 9.5. The enzyme retained more than 50% of
the initial activity after pre-incubation up to 24 h in buffers ranging
from pH 6.0–9.5 (Fig. 3b). The alkali-stability of CcXyl3A makes it
possible to collaborate with alkaline xylanase, this would be use-
ful in xylan degradation. The effect of temperature on the enzyme
activity was investigated at optimal pH, and the maximum activity
2
547 bp, encoded a predicted protein of 848 amino acids with theo-
retical molecular mass of 88.6 kDa and pI value of 4.92 (http://web.
expasy.org/compute pi/). Signal sequence of CcXyl3A was analyzed
by the SignalP4.1 server (http://www.cbs.dtu.dk/services/SignalP/
)
, the lack of a signal sequence suggests CcXyl3A is an intracellular
enzyme. Analysis of CcXyl3A with BlastP and Pfam identified the
protein as a member of glycoside hydrolases family 3. Similar to
some GH3 enzymes, a protein domain at the carboxyl-end portion
named fibronectin type III-like domain was found in CcXyl3A, the
function of which is still not well defined.
The multiple amino acid sequence alignment indicated CcXyl3A
had 77, 73, 69, and 60% identity to the putative GH3 enzymes
from Cellulomonas carbonis T26 (Genbank KGM11479), Isoptericola
variabilis 225 (Genbank AEG45495), Microbacterium hydrocar-
bonoxydans (Genbank WP 045258019), and Jonesia denitrificans
◦
was observed at 45 C (Fig. 4a), which was in accordance with other
GH3 multifunctional enzymes (50 C for BglX-V-Ara and RubGX1,
40 C for BgxA1) [11–13]. The thermostability of CcXyl3A was inves-
tigated ranging from 30 to 50 C at a constant pH of 8.5. As showed in
◦
◦
◦
(
Genbank WP 015772335), respectively.
CcXyl3A is the first enzyme which exhibits xylosidase,
Fig. 4b, approximate 71.7% of its activity remained after treatment
◦
at temperature at 35 C for 1 h, however, the activity significantly
◦
◦
arabinofuranosidase and glucosidase functionality in C. cel-
lulans. Previously, several GH3 bifunctional ␤-xylosidases/␤-
glucosidases and multifunctional ␤-xylosidases/␤-glucosidases/␣-
l-arabinofuranosidases from different sources were characterized
decreased above 40 C, and completely lost at 50 C after 15-min
incubation.
The effects of metal ions and reagents on the enzymatic activity
of CcXyl3A were investigated. As shown in Table 2, CcXyl3A activity
2+
2+
3+
[
11–13]. We also compared CcXyl3A with these previously pub-
was strongly inhibited by 5 mM Cu , Hg , Fe , and reagents SDS
and DTT. It is noticed that Mn2+ was reported to be an enzyme
enhancer for some ␤-xylosidases [20]. However, the activity of
lished enzymes (Fig. 1). The results indicated that CcXyl3A
exhibited low similarities to enzyme Bgxa1 from the rumen con-
tents of a grass/hay-fed dairy cow (AGN51317, 29% identity),
RubGX1 from yak rumen metagenome (ADD17009, 30% identity),
2+
CcXyl3A was strongly inhibited by Mn at the concentration of
5 mM. CcXyl3A was almost completely inhibited by Cu2+, how-
Table 1
Summary of purification of recombinant CcXyl3A.
Purification step
Volume (ml)
Total Protein (mg)a
Total activity (U)b
Specific activity (U/mg)
Recovery (%)
Purification (fold)
Crude enzyme extract
Ni sepharose fastflow column
15.0
12.5
33.5
10.0
63.65
44.2
1.90
4.42
100
69.4
1.0
2.33
a
Protein was quantified according to the Bradford method using bovine serum albumin (BSA) as standard.
The activity was reported as activity on pNP␤Xyl (40 mM).
b

6
8
Y. Yuan et al. / Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
Fig. 1. Multiple amino acid sequence alignment of CcXyl3A with previously reported GH3 multifunctional enzymes Bgxa1 (AGN51317), RubGX1 (ADD17009) and BglX-V-Ara
WP 010920890). Dark shading indicates highly conserved residues in all sequences, lighter shading indicated conserved residues in 3 of the 4 sequences. Alignment was
generated using Clustal Omega and formatted with Genedoc. Putative catalytic residues are labelled, in which N* as nucleophile and A* as acid/base catalyst.
(

Y. Yuan et al. / Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
69
ever, Cu2+ did not strongly affect the enzyme activity of BgxA1,
in which only 17.7% of the activities was lost at the concentration
of 5 mM [11]. At the concentration of 50 mM, most metal ions and
+
reagents we tested inhibited the activity of CcXyl3A. However, K
and Na+ significantly activated the enzymatic activity of CcXyl3A.
The enzyme activity of CcXyl3A was increased 132.2% and 132.6%,
respectively, in the presence of K+ and Na+ at concentrations of
5
0 mM.
Previously, Huy et al. reported the ␤-xylosidase activity of GH3
bifunctional xylosidase/arabinofuranosidase rPcXyl was inhibited
by xylose or arabinose at concentrations 10–100 mM, the inhibition
mechanism was deduced to be competitive by preventing recogni-
tion between enzyme and substrate [21]. To assess the effect of
sugars on CcXyl3A activity, we examined the ␤-xylosidase activ-
ity of CcXyl3A in the presence of various concentrations of glucose,
xylose and arabinose (Fig. 5). Similar to rPcXyl, xylose and arabinose
exhibited inhibition effect on CcXyl3A activity at concentrations of
1
0 mM or above. However, glucose exhibited no significant effect
on the ␤-xylosidase activity of CcXyl3A, which is different from that
of rPcXyl. At the concentration of 100 mM, the enzyme activity was
5
0.9 ± 1.2%, 94.7 ± 2.1% and 48.4 ± 1.6% with the presence of xylose,
glucose or arabinose, respectively.
3.4. Substrate specificity of CcXyl3A on different substrates
The specificity of CcXyl3A on various substrates was investi-
gated. Using p-NP compounds as substrates, the hydrolysis order
was pNP␤Xyl » pNP␣Araf > pNP␤Glc. Other pNP-␤-glycosides and
pNP-␣-glycosides we tested were not substrates for CcXyl3A
(Table 3). This result suggested that CcXyl3A was a multifunc-
tional ␤-xylosidase/␣-l-arabinofuranosidase/␤-glucosidase. Using
pNP␤Xyl as a substrate, the kinetic parameters of purified recom-
binant CcXyl3A were determined. The Km, Vmax, and kcat values
Fig. 2. SDS-PAGE analysis of recombinant CcXyl3A on 10% resolving gel. Lane 1, cul-
ture lysate of E. coli BL21-pET28a-ccxyl3a after IPTG induction; lane 2, supernatant
of the culture lysate of E. coli BL21-pET28a-ccxyl3a after IPTG induction; lane 3,
CcXyl3A purified from Ni sepharose fastflow column; M, molecular weight marker
−
1
were 4.0 ± 0.1 mM, 10.1 ± 0.4 ␮mol/min/mg, and 15.4 ± 0.1 s
,
respectively. The catalytic efficiency kcat/Km was determined to
−1
−1
. In previous reports, two multifunctional
be 3.85 ± 0.1 mM
s
(
PageRuler Prestained Protein Ladder, Thermo Scientific).
␤
-xylosidase/␤-glucosidase/␣-l-arabinofuranosidase BgxA1 and
BglX-V-Ara belonging to GH3 were characterized [11,13]. Both
BgxA1 and BglX-V-Ara were reported to show highest activity
on pNP␤Glc, followed by pNP␤Xyl and finally pNP␣Araf, which
was different from that of CcXyl3A. The catalysis efficiency of
gene ccbgl1a from C. cellulans was cloned and overexpressed in
E. coli [18]. Enzymatic analysis showed that CcBgl1A exhibited high-
est activity on pNP␤Glc, followed by pNP␤Xyl and finally pNP␣Araf,
−
1
−1
CcXyl3A (3.85 ± 0.1 mM
s for pNP␤Xyl) was higher than BgxA1
the hydrolysis order was different from that of CcXyl3A. The catal-
−1
−1
−1 −1
−1 −1
,
(
3.0 ± 0.3 mM
s
for pNP␤Xyl) and BglX-V-Ara (0.14 mM
s
ysis efficiency of CcBgl1A for pNP␤Xyl was 0.19 ± 0.02 mM
s
for pNP␤Xyl). In our previous study, a novel GH1 ␤-glucosidase
which was lower than that of CcXyl3A.
Fig. 3. Effect of pH on activity (a) and stability (b) of CcXyl3A using pNP␤Xyl as substrate. The optimal pH of CcXyl3A was determined ranging from pH 2.0–10.5 using
following buffers: sodium acetate buffer, pH 2.0–6.0; Na2HPO4-NaH2PO4 buffer, pH 6.0–8.0; Glycine-NaOH buffer, pH 8.0–10.5. The maximum activity obtained was defined
◦
as 100%. The pH stability of CcXyl3A was determined by pre-incubating CcXyl3A in different pH for 24 h at 4 C, then determining the percentage of residual activity under
standard assay conditions. The activity of CcXyl3A without pre-incubating was defined as 100%. Results are presented as means ± standard deviations (n = 3).

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Y. Yuan et al. / Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
Fig. 4. Effect of temperature on activity (a) and stability (b) of CcXyl3A using pNP␤Xyl as substrate. The optimal temperature was determined at different temperatures
◦
ranging from 20 to 90 C. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 1 h at different
temperatures. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations (n = 3).
Table 2
Effects of metal ions and chemical agents on the activity of CcXyl3A.
Metal ions or reagents
Relative activity (%)a
5
mM
50 mM
KCl
107.7 ± 2.2
108.0 ± 4.7
95.0 ± 3.3
106.3 ± 3.8
2.5 ± 0.1
132.2 ± 3.7
70.0 ± 3.8
20.3 ± 0.5
132.6 ± 2.4
0.4 ± 0.0
MgCl2
CaCl2
NaCl
CuCl2
FeCl3
HgCl2
BaCl2
MnCl2
DTT
b
21.4 ± 1.9
1.3 ± 0.0
–
–
78.0 ± 2.8
59.5 ± 4.4
56.2 ± 1.5
72.6 ± 1.1
6.3 ± 0.3
31.8 ± 2.8
16.0 ± 0.8
49.2 ± 1.1
1.2 ± 0.1
–
EDTA
SDS
a
The activity assayed in the absence of cations or reagents was taken as 100%.
Results are presented as means ± standard deviations (n = 3).
b
Fig. 5. Effect of sugar concentration on the ␤-xylosidase activity of recombinant
CcXyl3A. CcXyl3A was pre-incubated with respective concentrations (5, 10, 25, 50
and 100 mM) of each sugar in 100 ␮l of 50 mM Na2HPO4-NaH2PO4 buffer (pH 8.0)
for 1 h at 37 C. Then reactions were carried out by adding pNP␤Xyl to a final con-
centration of 40 mM for 10 min. The residual activity in the absence of sugar was
Not detected.
◦
(Table 3), but release xylose from xylobiose and xylotriose (Table 3
and Fig. 6a). When using a xylo-oligosaccharides mixture as the
substrate, the enzyme showed considerable hydrolytic activity. As
shown in Fig. 6b, the xylo-oligosaccharides mainly containing xylo-
biose, xylotetraose and xylohexaose were completely converted
after 10 min incubation, xylose was the sole product.
defined as 100%. Results are presented as means ± standard deviations (n = 3).
Table 3
a
Relative activity of CcXyl3A on different substrates.
Substrateb
Relative activity
Substrate
Relative activity
(%)
c
(%)
3.5. Synergistic effect between CcXyl3A and xylanase in
degradation of xylan
pNP␤Glc
pNP␤Xyl
pNP␤Man
pNP␤Gal
pNP␣Araf
pNP␣Glc
pNP␣Gal
oNP␤Glc
pNP␣Man
9.6 ± 0.2
xylobiose
xylotriose
cellobiose
cellotriose
sophorose
laminaribiose
laminaritriose
gentiobiose
100 ± 2.5
100 ± 2.4
0
100 ± 0.0
0
␤-Xylosidase catalyzes the breakdown of ␤-1,4-linked
1.4 ± 0.1
14.4 ± 2.1
1.5 ± 0.3
0.3 ± 0.1
6.4 ± 0.1
0.4 ± 0.1
0
xylooligosaccharides, which are produced from degradation
of xylan by xylanases. To examine the ability of CcXyl3A in the
saccharification of lignocellulosic xylan, the enzyme was incu-
bated with beechwood xylan on its own and in combination
with a commercial available xylanase. During the reaction, the
percentage of residual activity of CcXyl3A (pH 8.0 and 37 C) was
measured with pNP␤Xyl as a substrate. The result showed that
approximate 65% of initial hydrolytic activity was reserved after
19.9 ± 2.0
17.5 ± 0.8
14.4 ± 1.2
0
a
◦
◦
Reactions were performed with 40 mM substrate, pH 8.0, at 37 C for 10 min.
Absorption caused by released p-nitrophenol was measured at 405 nm. The
b
relative activity on pNP␤Xyl was defined as 100%.
c
The data are reported as means ± standard errors from the mean for three inde-
8
h incubation. During the hydrolysis process, the amount of
pendent experiments.
released reducing sugars was determined (Fig. 7a), the hydrolytic
products were analyzed by TLC (Fig. 7b). Although CcXyl3A com-
pletely hydrolyzed the xylooligosaccharides to xylose with good
activity, it showed very poor hydrolytic activity on beechwood
xylan. After incubated with CcXyl3A for 8 h, only a few amount
of xylose was released from beechwood xylan. The beechwood
xylan was partially degraded after incubated with xylanase for
Cello- and xylo-oligosaccharides are common moieties found
in plant cell walls, which could be used in biomass conversion for
biofuel production. Therefore, the hydrolytic activity of CcXyl3A
on cello- and xylo-oligosaccharides were determined. The results
showed that CcXyl3A had no activity on cello-oligosaccharides

Y. Yuan et al. / Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
71
Fig. 6. TLC and HPLC analysis of the hydrolysis products of xylo-oligosaccharides by CcXyl3A. (a) TLC analysis of the hydrolysis process of xylobiose by CcXyl3A. Std: standards
xylose (X1) and xylobiose (X2). (b) TLC analysis of the hydrolysis process of xylotriose by CcXyl3A. Std: standards xylose (X1), xylobiose (X2) and xylotriose (X3). (c) HPLC
analysis of the hydrolysis process of xylooligosaccharides by CcXyl3A. Curve 1, transformed substrate containing xylobiose (X2), xylotetraose (X4) and xylohexaose (X6);
curve 2, standard xylose (X1); curve 3, hydrolysis product.
8
h, with xylobiose and xylooligosaccharides as the main products.
functioned as a ␤-xylosidase in saccharification of xylans, which
promoted the xylan degradation by the synergistic action with
xylanase.
The combination of CcXyl3A and xylanase in the reaction mixture
significantly improved the liberation of the reducing sugars from
beechwood xylan. At the ratio of 1:1 (CcXyl3A/xylanase), the
amount of released reducing sugars was 1.7-fold relative to that
of endoxylanase alone. After 8-h incubation, most of the xylobiose
and xylooligosaccharides was hydrolyzed and a significant amount
of xylose was accumulated. These results suggested that CcXyl3A
4. Conclusions
In conclusion, we have identified, cloned and characterized
the ␤-xylosidase/␣-arabinosidase/␤-glucosidase CcXyl3A from

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Y. Yuan et al. / Journal of Molecular Catalysis B: Enzymatic 131 (2016) 65–72
Fig. 7. Time-course analysis of xylan hydrolysis by the combination of recombinant CcXyl3A and T. lanuginosus xylanase (Xyn). (a) The amount of released reducing sugars
during xylan hydrolysis were determined by DNS reagent; (b) The hydrolysis of xylan was analyzed by TLC method. STD: standards xylose and xylobiose, lane 0, 1, 2, 4, 8
means different reaction times.
C. cellulans sp. 21, which was the first multifunctional GH3
enzyme reported in this strain. CcXyl3A can effectively hydrolyze
xylooligosaccharides to release xylose and exhibited a synergis-
tic effect with endoxylanase in xylan degradation. The discovery
and characterization of the multifunctional ␤-xylosidase/␣-
arabinosidase/␤-glucosidase CcXyl3A would be helpful for the
application of C. cellulans in the saccharification of lignocellulosic
material.
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